A broad-range PCR technique for the diagnosis of infective endocarditis

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h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

A

broad-range

PCR

technique

for

the

diagnosis

of

infective

endocarditis

Ines

Boujelben

a

_,

_Radhouane

_Gdoura

b,∗

_,

_Adnane

_Hammami

a

a_Hospital_Sfax,_Department_of_Microbiology_and_Research_Laboratory_{“Microorganismes}_et_Pathologie_Humaine”_Habib_Bourguiba,_Sfax,

Tunisia

b_Université_de_Sfax,_Faculté_des_Sciences_de_Sfax,_Laboratoire_de_recherche_Toxicologie_{Microbiologie}_{Environnementale}_et_Santé

LR11ES06,Sfax,Tunisia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19March2016 Accepted23March2017 Availableonline9February2018 AssociateEditor:AfonsoBarth

Keywords: Broad-rangePCR Infectiveendocarditis Conventionalmethods Negativeculture Phenotypicinvestigations

a

b

s

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Infectiveendocarditis(IE)remainsasevereandpotentiallyfataldiseasedemanding sophis-ticateddiagnosticstrategiesfordetectionofthecausativemicroorganisms.Theaimofthe presentstudywastodevelopabroad-range16SribosomalRNAgenepolymerasechain reac-tionintheroutinediagnosticofIEfortheearlydiagnosisoffataldisease.Abroad-rangePCR techniquewasselectedandevaluatedintermsofitsefficiencyinthediagnosisof endocardi-tisusing19heartvalvesfrompatientsundergoingcardiovascularsurgeriesattheHabib BourguibaHospitalofSfax,Tunisia,onthegroundsofsuspectedIE.Theresults demon-stratedtheefficiencyofthistechniqueparticularlyincasesinvolvingalimitednumberof bacteriasinceithelpedtoincreasedetectionsensitivity.Thetechniqueprovedtobeefficient, particularly,inthebacteriologicaldiagnosisofIEincontextsinvolvingnegativeresultsfrom conventionalculturemethodsandothercontextsinvolvingbacterialspeciesthatwerenot amenabletoidentificationbyphenotypicinvestigations.Indeed,thesequencingofthe par-tial16SribosomalRNAgenerevealedthepresenceofBartonellahenselae,Enterobactersp.,and

Streptococcuspyogenesinthreeheartvalveswiththenegativeculture.Itshouldbenotedthat theresultsobtainedfromthepolymerasechainreaction-sequencingidentiﬁcationapplied totheheartvalveandthestrainisolatedfromthesametissuewerenotconsistentwiththe onesfoundbytheconventionalmicrobiologicalmethodsinthecaseofIEcausedbyGemella morbillorum.Infact,theresultsfromthemolecularidentiﬁcationrevealedthepresenceof

Lactobacillusjensenii.Overall,theresultshaverevealedthattheproposedmethodissensitive, reliableandmightopenpromisingopportunitiesfortheearlydiagnosisofIE.

∗ _{Corresponding}_author.

E-mail:[email protected](R.Gdoura). https://doi.org/10.1016/j.bjm.2017.03.019

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Introduction

Conventional bacteriologicaldiagnostic methods consist in the isolation and identiﬁcation of the causative agent of aninfectionfromapathologicalproduct.Thisidentiﬁcation is based on the study of the phenotypic traits (morpho-logical, cultural and biochemical) of the isolated bacteria. The microbiological diagnosis of infective endocarditis (IE) ismainlybasedonbloodculture,excisedcardiacvalve tis-sue,or infectedemboli.Theconventionalapproachforthe microbiologicaldiagnosisofIEissuccessfulin92–95%ofcases whereinamicroorganismispresent.Streptococciand Ente-rococciaccountfor45–60%ofthecasesofIE.Staphylococci representthesecondmostfrequentgroupassociatedwithIE. Variousfungiorbacteria,suchasEnterobacteriaceae,causethe remainingcasesofIE.Infact,somebacteriaaremore specif-ically associatedwith IE, including those from the HACEK group(Haemophilus,Aggregatibacter,Cardiobacterium,Eikenella

andKingella).1

Althoughconventionalculturesarenegativein5–8%ofIE cases, IEis a challengingdisease todiagnose. Thisis par-ticularly due to the wide variation in the criteria used to deﬁneitevenaftersurgery.Culturesandmicroscopymaynot revealthecausativeorganismintheinfectedvalve.Insuch cases,slow-growingornon-culturablemicrobesmay bethe etiologicagents,andthepatientmayhavereceived antimi-crobialtreatmentatthetimethespecimenwasobtained.2_In

addition,phenotypicidentiﬁcationtechniquesarenot appli-cableforbacteriaexpressinglittlediscriminatingphenotypic characteristics.Inrecentyears,researchershaveincreasingly become interested in the search for viable strategies and techniques to overcome the inadequacies and limitations associatedwiththeconventionalmicrobiologicalmethodsof IEdiagnosis.

Theliteratureindicatesthatculture-independent molec-ulartechniquesthat involvethe useofbroad-rangePCRto detectandamplifybacterialDNAandsequencingofthegenes codingfor16SrRNAmay openpromisingopportunitiesfor establishingtheetiologyoftheinfection.Infact,theuseof thesetechniqueshasrevolutionizedthedetectionand anal-ysisofinfectiousdiseasesandthewaystocombatthem.2,3

Furthermore, the literature indicates that the nested-PCR approachoffersarapid,efﬁcientandreliabletoolforthe detec-tionofbacterialinfectionsfromclinicalspecimens.4,5 _Some

studieshave,however,reportedthatthefrequentincidence offalse-positiveresultsis amajor problemhampering the applicationofnestedPCRsinlarge-scaleclinicalsettings.4,6

Accordingly,theliteraturehighlightstheimportanceofthe ampliﬁcationofpartofthe16SrRNAgeneanddirect sequenc-ing of the amplicon to identify microbes in valve tissues. Thedeterminedsequenceisoftencomparedtothereference sequences, a procedure that often allows for the identiﬁ-cationof the causative agent. Theresults of conventional bacteriologicalor/andhistopathologicaldiagnostic methods forIEhaveoftenbeencomparedtothoseobtainedbyPCR.1,2

Thebroad-rangePCRappearedarelativelyeasyandreliable methodthat gives accurate results whenapplied to surgi-callyremoved heart valvesofpatients withIEand may be usedasanadjunctforcaseswhereculturalmethods fail.2

The ampliﬁcation of the gene that encodes the 16S rRNA ofeubacterium-speciﬁcsequences from heartvalvetissues seemtoofferanewpromisingtoolfortheetiologicaldiagnosis ofIE.Thismethodhasallowedforthedetectionoffastidious organisms, suchasTropherymawhipplei,Coxiellaburnetiiand

Bartonellaspp,whichrequirespecialcultureconditions.1,2_The

broad-rangePCRtechniquehasalsoprovedefﬁcientfor con-ﬁrmingthepresenceofseveralmicroorganismsresponsible for IE,including Granulicatella elegans, Streptococci, Staphy-lococci,Enterobacter,Borreliaburgdorferi,Candidaalbicans,and

Aspergillusspecies.7,8_Lactobacilli_have_been_identiﬁed_in_some

clinicalreportsascausalagentsofIE,meningitisandseveral otherhealthdiseases.9

Consideringthegrowingglobalconcernsoverthe preva-lence of IE and the promising potential that molecular techniquesmightopenfortheidentiﬁcationandanalysisof thisseriousinfection,thepresentstudyaimedtoinvestigate thefeasibilityandpotentialofabroad-rangePCRtechnique, basedonbacterial16SrRNAgene,inthediagnosisofIE.

Materials

and

methods

Bacterialstrainsandspecimens

ThereferencestrainsusedascontrolorganismsinthePCR performed in this study were Escherichia coli ATCC 25922,

Staphylococcus aureus ATCC 43300, Streptococcus pneumoniae

ATCC 49619, Haemophilus inﬂuenzae ATCC 1399, Salmonella TyphimuriumWHO32,KlebsiellapneumoniaeWHO1, Enterococ-cusfaecalisATCC159,CitrobacterfreundiiWHO9,Pseudomonas aeruginosaWHO8,andSerratiamarcescensWHO10.Someother strainsthatposedidentiﬁcationproblemswerealsoisolated fromtheheartvalvesofthepatients,namelyAcinetobacter bau-mannii,Micrococcuslylae,andGemellamorbillorum.Theisolates ofeachoftheabovebacteriawereobtainedfromthe labora-toryofclinicalmicrobiologyintheHabibBourguibahospital ofSfax,Tunisia.

SuspensionsofE.coliATCC25922,S.aureusATCC43300, andS.pneumoniaeATCC49619containing(106_,₁₀5_,₁₀4_,₁₀3_,

102_and₁₀_CFU/mL)_were_also_prepared_by_routine_culture_and

dilutioncounts.10

Thestudy used atotalof19 heart valvesfrom patients whounderwentheartvalvereplacementsurgeries(Prosthetic valve) atthe cardiovascularservice oftheHabib Bourguiba hospitalofSfax,Tunisia,onthegroundsofsuspectedIE.

DNAextraction

The DNA of the bacterial strains of each specimen valve tissue was extracted by the Cetyltrimethylammonium Bromide–Phenol-Chloroform/Isoamyl Alcohol method ((CTAB)–Phenol-Chloroform/Isoamyl Alcohol). Prior to (CTAB)–Phenol-Chloroform/Isoamyl Alcohol extraction, 500␮L oflysis buffer (200mMNaCl, 20mM TrisHCl,pH 8, 50mMEDTA,pH8, and 1%SDS)and 25␮LofProteinase K (10mg/mL) (Sigma) were added to approximately 10mgof valvetissue.Themixturewasthenvigorouslyagitatedand incubated at 65◦C for 30min or until the complete disso-ciation of valve tissue fragments. The enzymatic reaction

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wasstoppedbyincubationat95◦Cfor10min,andsamples werecentrifugedat10000×gfor5s.Thesampleswerethen harvestedbycentrifugationat14000×gfor20min.

Afterhomogenization, thesamples wereincubated in a solutionof(CTAB)-NaCl(100␮Lof5MNaCland80␮Lof10% CTAB) for 10minat 65◦C, and then mixed with 750␮L of chloroform–isoamyl alcohol (24:1 [vol/vol]) and centrifuged for 15min at 14000×g in an Eppendorf centrifuge. The aqueousphasewasseparated,mixedwith750␮Lofphenol chloroform–isoamyl alcohol (25:24:1 [vol/vol/vol]), and cen-trifugedfor15minat14000×ginanEppendorfcentrifuge. The obtained aqueous phase was reseparated and mixed withanequalvolumeofisopropanol.Thesampleswereleft to rest for 1h at 80◦C and then centrifuged for 15min at 14000×g.TheDNApelletwaswashedwith70%ethanol, air-dried,anddissolvedinaﬁnalvolumeof100␮LofTEbuffer. TheDNAextractionforeachbacterialdilutionwasperformed using(CTAB)–Phenol-Chloroform/IsoamylAlcoholextraction andcommercialkits(QIAamp® DNAMiniKit)inaccordance withthemanufacturers’instructions.Tocomparetherelative efﬁcacyofthebothextractionmethods.

ThegenomicDNAoftheisolateswasextractedwiththe traditionalboilingmethodasdescribedbyHarrisetal.(2003). Inbrief,6bacterialcolonieswasre-suspendedin1mLofsterile salineandboiledfor10.

DNAconcentrationwasestimatedbygelmigration.

PretreatmentofTaqDNApolymerasetoremove contaminatingbacterialDNA

PriortothePCRampliﬁcation,apretreatmentprotocolwas developedto preventthe occurrenceof falsepositives due to the presence of Thermophilus aquaticus DNA in the Taq polymerase. In brief, the water, buffer, deoxynucleoside triphosphates, and Taq DNA polymerase components were mixedandincubatedfor60minat37◦Cwith1U,1.5Uand 2UofDNaseI(AmershamBiosciences).Theenzymewasthen inactivatedbyincubationat90◦Cfor30min,followedbythe additionofprimersandtemplateDNA.ThePCRampliﬁcation phasewasthenstarted.Thedifferentcomponentswerealso mixedandincubatedwith1UoftherestrictionenzymesSau 3AIandAluI.4,6

Broad-rangePCRampliﬁcationof16SrRNAgenesand sequencing

Initially,theextractedDNAwastestedtochecktheabsence ofPCRinhibitorsinthevalvetissuebytheampliﬁcationof 209bpfragmentofahuman␤-globingene.␤-Globinpositive sampleswerethenexaminedbybroad-rangePCR.11

ForthenestedPCR,primersdescribedbyWilkinsonetal.5

wereusedinthepresentstudy.

Theﬁrst-round PCR reaction was carried out ina reac-tion mixtureof50␮Lper tube containing10␮Lofl0× PCR buffer,0.4␮MofeachPCRprimers(NW9/NW17,NW9/NW12, NW11/NW17 or NW11/NW12), 0.2␮M of deoxynucleoside triphosphates,and0.0025UofTaqpolymerase.Two oligonu-cleotideprimerpairs,NW9(5-GCTAACTAACGTGCCAGCAG-3) andNW17(5-TAAGGGCCATGA(T/G)GA(C/T)TTGAC-3),were used.PCRwasperformedusingthe Gene-AmpPCRSystem

9700 (Perkin Elmer Cetus). The PCR protocol consisted of an initialcycle at 95◦C for 5min, followed by40 cycles of denaturationat94◦Cfor30s,annealingat50◦Cfor30s,and extensionat72◦Cfor45s,withafinalcycleat72◦Cfor7min. EachPCRrunincludedapositivecontrol(E.coli)and2 nega-tivecontrols(previousnegativelytestedsamplesanddistilled water).Thesecond-roundPCRwascarriedoutwiththesame volumeandreagentsmentionedaboveusingprimersNW11 (5-GAGGCCTACAAGCGGTGGAG(C/G)ATGTG-3)andNW12(5 -GAGGCCTCCAACATCTCACGACACGAG-3).Thethermocycling parameterswereidenticaltotheonesusedinthefirstround, exceptfortheannealing temperature,whichwasincreased to58◦C,and extensionstep,whichwas performedat72◦C for30s.Anamountof5␮LofDNAextractwasaddedinthe first-round PCR,and1␮LofthePCRproductwasaddedfor the reamplification round.Thepurified PCRproducts were sequenced with an ABI PRISM BigDye sequencing kit and analyzedwithanABI 3100-Avantgeneticanalyzer (Applied Biosystems).

Phylogeneticanalysis

Theprogram MolecularEvolutionary GeneticAnalysis soft-ware, ver.7.0(MEGA7.0; http://www.megasoftware.net)was performedinordertoeditandalignthesequenceﬁles,which weremanuallyadjusted.Inthisstudy,phylogenetictreewas generatedusingmaximumparsimony(MP)inMEGA5.0. Boot-strap values forthe maximum parsimony tree(MPT) were caculatedfor1000replicates.Theeditedfragment(155bp)of the16SrRNAsequenceswerecomparedwithotheravailable

LactobacillusspeciessequencesintheGenBank.Furthermore, thesequencesofsomeknownspeciesofLactobacilluswere downloadedfromGenBankandusedtoreconstruct16SrRNA regionphylogenetictrees.

PCR-RFLP

ThePCRproductsfromculturesandclinicalspecimenswere digestedusing10–17␮LofPCRampliﬁedDNArestrictedwith 1UofEcoRI,FokIandDdeIinatotalvolumeof20␮Laccording tothemanufacturer’sinstructions(Invitrogen)andincubated for4hat37◦C.Therestrictionfragmentswereseparatedby electrophoresisonagarosegel3%.

The search for recognition sites of restriction enzymes at the nucleic acid sequences was performed using the REBASEprogram(http://rebase.neb.com/rebase/rebase.html). Thisprogramhascommonlybeenusedfortheidentiﬁcation ofpredictedenzymesandestimationofthesizeofdigested ampliconfragments(Genbanksequences).

Results

and

discussion

ComparisonofDNAextractionmethods

Two different DNA extraction methods were examined to ascertaintheirrelativeeffectivenessforextractingbacterial DNA from gram positive and negative bacteria. The CTAB methodwasthemosteffectiveforthedetectionofDNA pre-paredfrom thepurebacterialcultureofGram-positive and

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1 2 3 4 5 ₁ ₂ ₃ ₄

A

B

710 bp 155 bp 7 8 6

Fig.1–Nested-PCRusingtheprimerpairsMW9/NW17andNW11/NW12.(A)First-roundPCRampliﬁcationwithprimer pairsMW9/NW17.Lane1DNAladder;lane2–7containeddifferentsuspensionsofStreptococcuspneumoniae:lane2– 106_CFU/mL;_lane₃_–₁₀5_CFU/mL;_lane₄_–₁₀4_CFU/mL;_lane₅_–₁₀3_CFU/mL;_lane₆_–₁₀2_CFU/mL;_lane₇_–₁₀_CFU/mL;_lane₈_–

negativecontrol.(B)Second-roundofPCRampliﬁcationwithprimerpairsNW11/NW12.Lane1DNAladder;lane2,3 contained1␮Lfromtheﬁrst-roundPCR;lane2–suspension102_CFU/mL_of_{Streptococcus}_pneumoniae;_lane₃_–_suspension

10CFU/mLofStreptococcuspneumoniae;lane4contained1␮Lofthenegativereactionmixturefromthefirstround.(C) Second-roundofPCRamplificationofthenegativereactionmixturewithprimerpairsNW11/NW12.Lane1DNAladder; lane2–6contained1␮Lofthenegativereactionmixturefromthefirstround;lane2negativereactionmixturewithout pretreatment;lane3–6negativereactionmixturetreatedwith1U,1.25U,1.5Uand2UofDNaseI,respectively.

negativebacteria(positivitywasobservedtillthesuspension 102_CFU/mL_for_S._pneumoniae_(Fig._1A)_and ₁₀_CFU/mL_for_S.

aureusandE.coli).Althoughthekitmethodwaslesseffective intheextractionofbacterialDNA,positivitywasobservedtill thesuspension104_CFU/mL_for_all_the_tested_strains.

TheliteratureindicatesthattheextractionofbacterialDNA could beaccomplished withdifferent degreesofefﬁciency, depending on the method used.13,14 _In _the _present _study,

theapplicationofthein-housesamplepreparationmethods showedhigherlevelsofefﬁciency.Infact,thecaseinvolving manipulationfrom complex matrices, suchaspathological products,suggestedthatthesamplescouldpresumably con-tainvariousinhibitorsthatneedtobeeliminatedbeforethe PCR.15_Accordingly,_several_studies,_have_previously_reported

ontheextractionefﬁciencyofCTAB,anditspromising poten-tialfortheenhancementofPCRsensitivity.11,16,17

Validatonofthebroad-rangePCRonthereferencestrains

Theintroduction of the broad-rangePCR inthe laboratory ofclinical bacteriology requires its validation on reference strains beforeits application on bacterialstrains of uncer-tainidentiﬁcationorpathologicalproducts.12 _To_ensure_the

speciﬁcity ofthe broad-range PCR,the PCRproducts of10 referencestrains were sequenced usingthe primers NW11 (fragmentlength of263bp) and NW12 (fragmentlength of 584bp).Thedeterminationofthesetwosequences allowed forthedetectionofthesequenceofaregionof155bp.The sequences of the three different fragments(263bp, 584bp, 155bp)ofthe16S rRNAgeneofeachreference strainwere comparedusingtheBLASTprogram.Theresultsrevealedthat thereferencestrainunderinvestigationshowedhighlevels ofsequenceidentity.Theresultsobtainedforthesequence data on the molecular identiﬁcation of bacteria of these strainsusingasmallfragment(155bp)ofthe16SrRNAgene

conﬁrmedthediscriminatingpotentialofthebroad-rangePCR technique.

ApplicationofnestedPCRtechnique

The efﬁciency ofthe nested PCR technique was evaluated using three bacterial suspensions of the three reference strains (S. aureus, S. pneumoniae and E. coli) containing a decreasing number of bacteria (106_, ₁₀5_, ₁₀4_, ₁₀3_, ₁₀2 _and

10CFU/mL).TheamplificationoftheseextractedDNAusing the universalprimers NW9/NW17duringthefirst roundof PCRallowedfortheamplificationofthe16SrRNAgeneofa bacterial suspensioncontaining≥102CFU/mL(Fig.1A).The second PCR using the universal primers NW11/NW12 was appliedtoabacterialsuspensioncontaininganumberof bac-teria≤102_CFU/mL._The_16S_rRNAs_of₁₅₅_bp_were_amplified

intheexpectedsizeandwithhighintensitiesforthe differ-entbacterialsuspensionstested(Fig.1B).However,theresults showedthepresenceofanamplificationwiththesamesizeof thepositivecontrolandwithhighintensityforallthenegative controlsafterthesecond-roundofPCR(Fig.1B).No amplifica-tionofthistypewasobservedinthefirst-roundPCR.Asimilar resultwaspreviouslyreportedbyCarrolletal.6_who_detected

ampliﬁcationinthenegativecontrolofthereactionafterthe nestedPCR.

Thetechniqueofthe nested PCRtargetingthe16SrRNA geneusinguniversal andspecies-speciﬁc primershasbeen performed in several studies.3–5,12 _The _technique _has _also

been usedinvarious casesoflowabundanceorganismsto increasesensitivity.5,6,13_The_two-step_process_of_the_nested

PCRtechniqueresultedinthedilutionofpotentialinhibitors andprovisionofsufﬁcienttemplateforthesecondary reac-tion.Almostallthesamplesthatgavenegativeresultsinthe primaryPCRyieldedintoaspeciﬁcproductafternestedPCR, indicatingthepresenceofbacterialDNA.13

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However, the present results suggest that despite the improvedsensitivityachievedbytheapplicationofthenested PCR,thesecondamplificationcouldincreasetheriskoffalse positiveoccurrences.Manystudieshavereportedthatunlike thecasewithspecies-specificPCR,thevalidityofbroad-range PCRtechniquesfortheamplificationofthe16SrRNAgeneof broadspectrumofpathogenicbacteriaiscompromisedbythe highrateoffalsepositiveresults.18,19 _This_method_is_more

vulnerabletocontaminationthanspecies-speciﬁc PCR.20_In

the nested PCR, the second-round PCR performed on the ampliﬁcationproductfromapreviousPCRmayinvolve con-tamination,thusincreasingtherateoffalsepositives.4,6Such DNA contamination can occur due to improper manipula-tions(personnel,equipment,reagents,etc.)orduringvarious experimental phases (extracting DNA samples or prepara-tionofPCRreactionmixture).5,14,21_In_the_present _study,_all

necessary precautions were taken to avoid the emergence ofpotential contamination due to handling or operational mistakes. The different steps of bacterial DNA extraction, reaction mixture preparation, and ampliﬁcation execution were performed inseparaterooms as previouslydescribed elsewhere.20–22 _Furthermore, _all _reagents _and _equipments

usedinthePCRexperimentswereregularlydecontaminated beforeuse(pipettes,tips,sterilewater,buffer,etc.).The prepa-rationofthereactionmixtureanddepositionoftheextracted DNAwere carriedout inalaminarﬂowhoodundersterile areas.Nevertheless,anddespitealltheseprecautions,an inci-denceofcontaminationfromexogenousbacterialDNAwas observedduringthesecond-roundofthenestedPCR.Infact, numerousstudieshavereportedthattheTaqpolymeraseis themajorsourceofcontaminationwithexogenousbacterial DNA(Hughesetal.,1994;RandandHouck,1990).23_The

litera-turepresentsseveralmethodsofcontaminatedDNAremoval toavoidthis problem.6,18,19 _In _addition _to_the _proper

labo-ratoryorganizationandadequateseparationofthedifferent PCRstages21_the_techniques_proposed_for_the_elimination_of

contaminationbyexogenousbacterialDNAincludethe UV irradiationofthedifferentgradientsinthereactionmixture, andthepretreatmentofthe reactionmixturebyrestriction enzymesorDNaseI.6,18,23_The_long_UV_exposure_technique

has, however, been reported to reduce the activity of Taq polymerase.23

PretreatmentwithrestrictionenzymesandwithDNaseI

Theresultsrecordedafterthetreatmentofthereaction mix-turewithahighconcentrationofthetworestrictionenzymes (Alu I and Sau 3AI) revealed the absence of ampliﬁcation forall samples(negative and positive controls).Thiscould be attributedto the presenceof a high salt concentration by the uploading buffer of the restriction enzyme, which, inturn,couldhaveinduced theinhibitionoftheTaq poly-merase.Manystudieshavereportedthatthepretreatmentof thereactionmixturebeforetheadditionofDNAtemplatewith lowconcentrationsofrestrictionenzymesfailedtoeliminate contamination.Thisfailurecouldbeascribedtotheabsence ofcleavagesitesforbothrestrictionenzymesatthe exoge-nousDNAduetotheunknownsourceofthecontaminating

DNA,sincetheTaqpolymerasemaycontainDNAofunknown origin.23,24

The negative reaction mixturewas pretreated with dif-ferent concentrations of DNase I and exposed to various incubationperiodsduringthefirstandsecond-roundofthe PCR. Thispretreatmentallowedforthe eliminationof con-taminationcausedbyexogenousbacterialDNA.Theabsence ofamplificationinthenegativecontrol(water andreaction mixture)wasobservedafterthepretreatmentwithDNaseI at a concentration of1.5U and after anincubation period of 60min at 37◦C and 30min at 90◦C. The DNase I pre-treatmentprocess wasdevelopedbyHeiningeretal.18 who assayedDNaseIatdifferentconcentrationsandreportedon itsefficiencyintheeliminationoffalsepositiveresults.The concentrationrequiredforDNaseItoremovetheexogenous DNAoftheTaqpolymerasedependedonthenatureoftheTaq polymerase(Go-Taqpolymerase,DeepVentExo-polymerase, super-Taq polymerase,Ampli-Taqpolymerase,etc.)and the levelofenzymecontamination.18,19_The_cloning_and

sequenc-ingofthePCRfragmentproducedbyPromegaTaqpolymerase revealedthepresenceofatleast5differentclones,conﬁrming thattheTaqpolymerasecontainedtheunknownDNAfrom morethanonebacterialspecies.25

ThenestedPCRwasapplied(theprimerpairsNW9/NW17 fortheﬁrst-roundPCR,andNW11/NW12forthesecond-round PCR)afterthepretreatmentofthereactionmixturewith1.5U ofDNase I(60minat37◦C and for30minat90◦C) andby addingdifferentbacterialsuspensionsofS.pneumoniae(106, 105_, ₁₀4_,₁₀3_, ₁₀2 _and ₁₀_CFU/mL)._The_results_revealed_PCR

positivityonlyforthe suspensionscontaininganumberof bacteria≥103_CFU/mL. _Thus,_the_results_obtained_by_nested

PCRafterreactionmixturepretreatmentwithDNaseIatthe firstandsecond-roundsofPCRrevealedthatthePCRefficiency decreasedwhencomparedtotheoneobservedforthe broad-range PCR using the primer NW9/NW17 without reaction mixturepretreatmentwithDNaseI.Infact,thebroad-range PCRallowedfortheamplificationofbacterialsuspensionsofS. aureusandE.colicontaining10CFU/mL.Thepresentstudyhas shownthatthepretreatmentofthereactionmixturebyDNase ImaydamagetheactivityoftheTaqpolymeraseand, there-fore, inducethe occurrenceoffalsenegative results.Thus, despiteofferingtheadvantageofeliminatingcontaminated DNAinTaqDNApolymerase,thepretreatmentofthe reac-tionmixturebytheDNaseImayreducePCRsensitivity.The decreaseinPCRsensitivitycouldpresumablybeattributedto theexposureofTaqDNApolymerasetoelevatedtemperatures foralongperiodoftime.23_Accordingly,_the_optimization_of

DNaseItreatmentefﬁciencyrequirestheacquisitionofthe mostdurabletaqpolymeraseavailable.23_Overall,_and_in_spite

of its limitedability tofully denature the enzymewithout compromising Taq polymerase activity, DNase I pretreat-mentseemstobethemosteffectivecontaminationremoval method.23

Effectofampliconsizeonbroad-rangePCRsensitivity

ToinvestigatethepotentialenhancementofPCRsensitivity, DNA ampliﬁcationbybroad-rangePCRwasfurtherassayed using4primerpairs,namelyNW9/NW17(amplifyaregionof

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710bp),NW9/NW12(amplifyaregionof600bp),NW11/NW17 (amplifyaregionof280bp),andNW11/NW12(amplifyaregion of 155bp). The amplification of the DNA extract from the reference S. pneumoniae strain using these 4 primer pairs revealed amplification in the expected size. The intensity ofthePCRfragmentwasnotedtoincreasewiththedecreaseof ampliconsize.Themostintensebandwasobservedwiththe NW11/NW12primerpairs,whichamplifiedaregionof155bp. Infact,theshorterthebacterialstudiedfragmentswere,the higherthepossibilityofbacterialidentificationincases involv-ingasmallnumberofbacteriabecame.

Theﬁrststudy touse broad-rangePCRtargetingthefull 16SrRNAgeneforthedetectionofbacterialDNAinsynovial tissuesusedautomatedDNAextractiontechniques,which dif-fersfromthe in-housesamplepreparationmethodusedin thepresent study.26 _In_fact, _the_T4_Gene₃₂ _Protein_should

alsobeadded tothe PCRmix.Althoughthelasttechnique provedefficient,itsapplicationiscostly,time-consumingand high-expertise requiring. The diagnostic application ofthe broad-rangeamplification–sequencingmethodrequires ade-quatedatabaseswithgoodqualityandfull-lengthsequences of clinically important bacteria to allow genus or species identificationofbacterialpathogens,eitherdirectlyfrom clin-icalspecimensor from cultures.2 _Although_long_amplicons

can provide more information, they are more difﬁcult to generate, which results in the loss of sensitivity.3 _Longer

targets (e.g., primers 27f (forward)/1492r (reverse)) may be used when bacteria are abundant in the samples.28

How-ever,Schuurmanetal.20_used_the_primer_pairs_that_ampliﬁed

a 1500bp fragment of 16S r RNA gene and another step of reamplification to increase the sensitivity of the tech-nique.The identification bybroad-range PCRamplification andpartialsequencing ofthe 16S rRNAgenedirectly from clinical specimen and culture was previously reported in several studies proving the efficiency of the method to diagnoseIE.1,2,22,27,29

Contributionofthebroad-rangePCRtothediagnosisofIE

Thepresent study examined 19heart valvesfrom patients whounderwentvalvereplacementsurgeriesatthe cardiovas-cularserviceoftheHabibBourguibahospitalofSfax,Tunisia, onthegroundsofsuspectedIE.Theconventional bacteriolog-icalexaminationoftheheartvalvesshowedthepresenceof3 positivecases:onecaseofA.baumannii,acaseofG. morbillo-rum,andanothercaseofM.lylae.DNAextractionwasfollowed bytheamplificationofa209bpfragmentofthehumangene encoding␤-globin.Theresultsshowedtheabsenceof ampli-ficationexceptforoneheartvalve.Thiscouldbeexplainedby thepresenceofPCRinhibitors.Theamplificationofthegene forhuman␤-globinwasusedasacontroltoverifytheabsence ofPCRinhibitorsintheDNAextractsfromclinicalsamples.1,21

Toinvestigatethecontributionofthebroad-rangePCRfor thediagnosisofIE,thebacterial16SrRNAgenewas ampli-ﬁed using primer pairs NW11/NW12 from 18 heart valves thathadpreviouslyshowntheabsenceofPCRinhibitors.The results revealed ampliﬁcations in the expected sizes for 5 heartvalves.Thecomparisonbetweenthe resultsobtained by PCR tothose from the culture valvesrevealed that the broad-rangePCRshowedahigherlevelofpositivity(Table1). Moreover,threeheartvalvesthathadbeenfoundnegativeby conventionalbacteriologicalexaminationwerelabeled posi-tivebybroad-rangePCR.Twocaseswereconsideredpositive bybothconventionalmicrobiologicalmethodandbroad-range PCRtechnique.Theresultsalsorevealed aunique instance whereinaheartvalvewaslabeledasnegativebythe broad-range PCR but positive by the culture and ␤-globin PCR techniques.Ourresultwassimilartothosereportedby Gaudu-chonet al.1 _and_Vondracek_et _al.29_The_negative_PCR_result

in this case may have been dueto three factors:the long intervalbetweenantibiotictreatmentandsurgery;theuseof aninappropriatevalvefragmentforPCR(theactualinfected siteoftheheartvalvewasnotanalyzed)orthepresenceof

Table1–Resultsofbroad-rangebacterialPCRforheartvalvesandstrainisolatedfromthesametissue. Numberofheart

valvestested

Valveculture results

PCRandsequencing fromthevalve

Identity(%) PCRandsequencing fromtheisolatedstrain ofthevalve

Identity (%)

1 Micrococcuslylae Negative – Kytococcusschroeteri strain11356-2(JF514889)

98 1 Gemellamorbillorum Lactobacillusjensenii

strainA084(KF740715)

92 Lactobacillusjensenii strainA084(KF740715)

96 1 Acinetobacterbaumannii Acinetobactersp.

ESBL506B15133A (KJ831554) Acinetobacterbaumannii strain3H7a(KJ801666) 98 Acinetobactersp. ESBL506B15133A (KJ831554) Acinetobacterbaumannii strain3H7a(KJ801666) 96

1 Negative Bartonellahenselae isolateBM1374165 (HG726042)

91 Nogrowth –

1 Negative Streptococcuspyogenes strainMHW3(KJ620886)

78 Nogrowth –

1 Negative Enterobactersp.COR260 (KF447985)

96 Nogrowth –

12 Negative Negative – Negative –

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57 32 77 51 29 46 63 35 92 100 46

73 Isolate lactobacillus jensenii NW11

Valve lactobacillus jensenii NW11

Lactobacillus jensenii ATCC25258 (AF243176) Lactobacillus johnsonii (AJ002515)

Lactobacillus intestinalis (AJ306299) Lactobacillus kalixensis (AY253657)

Lactobacillus psittaci (AJ272391)

Lactobacillus delbrueckii (M58814) Lactobacillus amylovorus (M58805)

Lactobacillus amylolyticus (Y17361)

Lactobacillus hamsteri DSM 5661T (AJ306298) Lactobacillus crispatus (Y17362)

Lactobacillus gasseri (M58820)

Lactobacillus iners CCUG 28746T (Y16329)

Lactobacillus jensenii A084 (KF740715)

0.01

Fig.2–16SrRNAgene-basedphylogeneticdendrogramshowingthephylogeneticpositionofthestrainandthevalve relatedtoLactobacillusjensenii.Numbersatthenodesarebootstrapvalues.Bar,1%sequencedivergence.

PCRinhibitors(althoughextractionandampliﬁcationcontrols gavetheexpectedresults,andampliﬁcationofthebeta-globin genewaspositive).1,29

Theidentificationofthebacteriadetectedinthefive pos-itive heart valvesbybroad-rangePCR wasperformed after sequencing, usingthe sense primer NW11,and alignment, usingtheBLASTprogram(BasicLocalAlignmentSearchTool). Thesequencingofthepartial16SrRNAgenerevealedthe pres-ence ofBartonella henselae, Enterobacter sp,and Streptococcus pyogenesinthreeheartvalvesthatwerenegative(Table1).The negativityofcultureinthecaseofBartonellacouldbedueto thedifficultyofisolatingthebacteriabyconventional micro-biologicalmethods.Hadfieldetal.30_reported_on_the_first_case

ofendocarditiscausedbyB.henselaeusinganapproach simi-lartotheonedescribedinthisstudy.Severalstudieshavealso usedthistechniquetodetectBartonellaintheaorticvalveof patientswithculture-negativeIE.31,32_The_ﬁnding_of_two_heart

valvestobeculturenegativebutPCRpositiveinthecasesof

StreptococcusandEnterobacterwaspreviouslyattributedtoprior antimicrobialtreatmentofpatients.2,22 _Various_studies_have

alsoreportedcasesofendocarditisduetoStreptococcusand

Enterobacter.8,33

The PCR based methods are rapid, reliable and highly sensitive tools for the microbiological diagnosis of culture negativeIE.Thismethodcansubstantiallyimprovethe diag-nostic outcome of microbiological examination of excised heart valves not only for fastidious, slow growing,and/or non-culturablemicroorganisms but alsoforeasy-to-culture pathogens,suchasstreptococciandstaphylococci.1,8,27 _The

describedmolecularapproachprovedtobesuperiorto bacte-rialculture.1,2,22,27,29

Inthe present study,a good concordancewas observed betweenPCR and conventional bacteriological diagnosis in the case of IE caused by A. baumannii. Ryczak et al.34

reported that this bacterium was responsible for IE. The application ofbroad-rangePCR andsequencing showedan agreement between the sequence amplifiedfrom the DNA extractedfromthestrainisolatedandtheoneextractedfrom the tissuefrom thesame heart valve.However,theresults obtainedfromthePCRsequencingidentificationfromtheDNA extracted,fromaheartvalvewasnotconsistentwiththeone foundbytheconventionalmicrobiologicalmethodsinthecase ofIEcausedbyG.morbillorum.Infact,themolecular identifica-tiontechniquesrevealedthepresenceofLactobacillusjensenii

(Table1),whichhasalsobeendescribedbymanystudies.35,36

ToconfirmtheresultsfromPCRappliedtotheDNAextracted fromtheheartvalve,thepresentstudyoptedforthe identifica-tionofthestrainisolatedfromthesamevalve.Thesequencing oftheamplified16SrRNAgenefromtheisolatedstrain(G. mor-billorum) indicated (96%similarity toL.jensenii) that thestrain wasL.jensenii.Atreewasconstructedbytheneighbor-joining methodbasedonextractedDNAfromisolatedstrainandthe onefromtheheartvalve,showingthephylogeneticposition ofthestrainandthevalverelatedtoL.jensenii(Fig.2).The phylogenetictreeanalysisclearlydemonstratedthatthe16S rRNAofthestrainandthevalvewerelocalizedwithintheL. jenseniiclade.

Thisbroad-rangePCR,therefore,representsahighly accu-rate and versatile method for bacterial identification even whenthespeciesinquestionarenotoriouslydifficultto iden-tifybyphenotypic means.Theliteraturehasconfirmedthe importance of 16S rRNA amplification and sequencing as compared to the conventional microbiological methods in the identification of microorganisms with uncertain char-acterization by phenotypic tests.1,36,37 _However, _Woo _and

colleagues38,39_have_proven_that_the_{identiﬁcation}_of

Lactobacil-lusandGemellaspeciesbyculturemethodsisinsufﬁcientand thatthePCRofthe16SrRNAgene,followedbysequencing,is

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themethodofchoicefortheidentiﬁcationofsuch microorgan-isms.Wooetal.39_have_also_reported_that_the_species_of_Gemella

areimportantcausalagentsofIE.Forthepatientwithnegative PCRandpositivecultures,thestrainisolatedfromvalveswas identifiedasM.lylaebyconventionalmicrobiologicalmethods suchasID32STAPHgallery.Toconfirmthebacterial identifica-tion,theanalysisproceededbyamplifyingandsequencinga partofthe16SrRNAgeneofthisstrain.ThePCRresults indi-catedthatthe strainwasKytococcusschroeteri (Table 1).Our resultswereinaccordancewithwhatwaspreviouslyreported byMnifetal.40_They_have_reported_that_biochemical

identiﬁ-cationdoesnotdifferentiatebetweenM.lylaeandK.schroeteri

andthatfurthermolecularstudiesareneededtoachievemore identiﬁcationthatisaccurate.Beckeretal.41,42_reported_cases

ofendocarditiscausedbyK.schroeteri.Theseauthorshave con-firmedthatbiochemicalidentificationdoesnotdifferentiate betweenM.lylaeandK.schroeteriandthatfurthermolecular studiesareneededtoachievemoreaccurateidentification.

The sequences of the 16S rRNA gene of the isolates identiﬁedinthepresentstudy exhibitedabout97%of simi-laritytoknownsequencesavailabledatabase,whichprovided theeventual genusidentiﬁcation.Goldenbergeret al.2

pre-viously reported that, during their study,several reference sequences in the databases often presented only par-tialsequences, which isconsistent withthe results ofthe present study. However, these authors suggested that, in some cases, the 16S rRNA gene sequence analysis cannot beemployedasthesoletechniqueforspeciesidentification. Nestedprimersthatspanuniquesequencesinthe16SrRNA genein staphylococciand streptococci atthe specieslevel (species-specificPCR)were used, andtheir applicationwas justifiedbytheneedtoidentifythemostcommonly antici-patedspecies.3,27_PCR-RFLP_was_developed_for_the_detection

andidentiﬁcationatthespecieslevelofBartonellainclinical specimens.43

PCR-RFLP

Further tests based on the PCR-RFLP technique were per-formed to verify the specificity of the amplification and confirm the findings obtained by the identification via sequencingfromtheheartvalveandthestrainisolatedfrom thesamematerial.TheREBASEprogramwasusedtosearch forrecognitionsitesofrestrictionenzymesinthenucleicacid sequence.ToconfirmthecaseoftheIEwithL.jensenii,the PCRproductof710bpofthestrainisolatedandidentifiedas

G.morbillorumbasedonthe culturewasdigestedbyEcoRI. Thisrestrictionenzymecutonetimethesequenceof710bp ofthe16SrRNAgeneofG.morbillorumanddidnotdigestthe sameamplifiedregioncomingfromL.jensenii.Theobtained resultsdidnotshowarestrictionsiteintheamplifiedregion, confirmingthatthedetectedbacteriumwasL.jensenii(Fig.3A). Likewise,theuseofFokIandDdeIrestrictionenzymeswhich cutonetimethe PCRproducts of155bp forL.jensenii,but notthat ofG. morbillorum,willprovidefurthersupportthat thedetectedstrainwasaL.jenseniiandnotG. morbillorum.

Inthepresentwork,thePCRproductof155bpobtainedfrom theheartvalveofapatientoperatedforsuspicionofIEwithG. morbillorumwassubmittedtodigestionwithFokIandDdeI. Theresultsshowedarestrictedfragmentintheexpectedsizes

1 2 3 1 2 3 4 710 bp 155 bp 105 bp 76 bp 48 bp

A

B

Fig.3–PCR-RFLPfromtheheartvalveandthestrain isolatedfrompatientswithsuspectedinfectiveendocarditis withtheenzymesEcoRI,FokIandDdeI.(A)PCRproduct ampliﬁedfromtheisolatedstrain;lane1DNAladder;lane 2PCRproductnon-digested;lane3PCRproductdigested withtheenzymeEcoRI.(B)PCRproductampliﬁedfromthe heartvalve;lane1DNAladder;lane2PCRproduct

non-digested;lane3PCRproductdigestedwiththeenzyme FokI;lane4PCRproductdigestedwiththeenzymeDdeI.

(Fig.3B)of105bpand48bpafterdigestionwithFokIandtwo fragmentsof76bpafterdigestionwithDdeI.

FurtherassaysbasedonthePCR-RFLPtechniquewere per-formedtoconfirmthe caseofIE withB.henselaeidentified afterPCRandsequencingoftissuesfromvalveswithnegative culture.TherestrictionenzymeSau3AIcuttwofoldsinthe regionof155bpofB.henselae.ThedigestionofthePCRproduct (155bp)withSau3AIshowedthreefragmentsintheexpected size (66bp,53bp,39bp). Sau3AI did notcut the regionof 155bpofBartonellaquintana,thusestablishingB.henselaeas the eventualinfecting speciesinthis case.Theimplication oftheBartonellaspeciesinavarietyofhumandiseasesand thedifficultyofitsisolationfromclinicalspecimenshighlight theneedforthedevelopmentofbetterdetectionand iden-tificationmethods.43_In_fact,_both_B._quintana_and_B._henselae

werefoundtocauseendocarditisandpreviouslyidentiﬁedby PCR-RFLP.32,43_This_assay_might_open_new_promising

opportu-nitiesfortheearlydiagnosisofIEandaccuratedifferentiation betweenspecies.43

Conclusion

The present study has conﬁrmed that the broad-range ampliﬁcation–sequencing method allowedfor the achieve-ment ofamoresensitiveclinical diagnosisofendocarditis. ThisPCRmethodprovedtobesuperiortoconventional meth-odsindetectingbacteriafromheartvalves.Theapplication ofthismoleculartechnique,especiallyinthecaseof nega-tiveresults,substantiallyimprovedthediagnosisofnotonly highlyfastidiousandslow-growingmicroorganismsbutalso pathogensthataregenerallyconsideredeasytocultivate.In addition,thebroad-rangePCRshowedtobeeffectiveinthe

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identiﬁcationofmicroorganisms whosecharacterizationby phenotypictestswasuncertain.ThePCRtechniqueproposed inthisstudy alsooffersasimple,user-friendly,time-saving andcost-effectivetoolfortheidentiﬁcationofIE.Thus,this techniquemaybeapromisingtoolforapplicationinthe rou-tinetestingofinfectiousdiseases.

Conﬂicts

of

interest

Theauthorsconﬁrmthattherearenotconﬂictofinterestfor thisarticle.

Acknowledgements

TheauthorswouldliketoexpresstheirsinceregratitudetoMr. AnouarSmaouiandMrs.HanenBenSalemfromtheEnglish LanguageUnitattheFacultyofScienceofSfaxfortheir valu-ableandlanguagepolishingandeditingservices.

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